Adenosine is an important extracellular messenger in the heart. During periods of increased oxygen demand or decreased oxygen supply (hypoxia, ischemia), release of adenosine results in vasodilation as well as negative inotropic and chronotropic effects. These actions return the oxygen supply/demand ratio back towards normal. The overall objective of this project is to determine the mechanisms regulating adenosine formation by the heart. The major hypothesis to be tested is that receptor activation is an important regulator of adenosine formation. One specific hypothesis is that adenosine limits its own formation by activation of cardiac A1 receptors. This is proposed to occur via decreased cyclic AMP concentration and a resulting decrease in the activity of cytosolic 5e-nucleotidase. Another specific hypothesis is that alpha-adrenergic receptor activation increases cardiac adenosine formation by activating the phospholipase C second messenger system. Both of these effects on adenosine formation are proposed to be independent of any effects on cardiac energetics. Accordingly, experiments will be done in preparations that are less metabolically active than the beating heart. We will begin by using arrested heart preparations. We will also explore the use of dispersed or cultured cell preparations. In all cases cellular nucleotide concentrations will be measured along with adenosine release in order to separate adenosine formation driven by nucleotide degradation from receptor-mediated adenosine formation. We will use 31P-NMR for this purpose whenever possible.